Abstract Epidemiological and clinical studies have found that cholesterol gallstones are more prevalent in women than in men at all ages in every population studied. Accumulated evidence shows that the use of oral contraceptives and conjugated estrogens in women significantly increases the prevalence of gallstones. Estrogen therapy to men with prostatic cancer also leads to similar lithogenic effects. These findings clearly demonstrate that the increased risk of developing gallstones in women compared to men is related to differences in how the liver metabolizes cholesterol in response to estrogen. Our published studies have established a critical role for estrogen in enhancing cholelithogenesis by activating the classical estrogen receptor ? (ER?), but not ER? in the liver. However, the mechanisms mediating estrogen?s lithogenic actions on gallstone formation have become more complicated with the identification of a novel estrogen receptor, the G protein-coupled receptor 30 (GPR30). Our genetic findings support the candidacy of GPR30 as a compelling gene underlying a new gallstone gene Lith18. However, identifying the lithogenic mechanisms of GPR30 has been a focal point of interest because it is still unknown whether GPR30 plays a major role in estrogen-induced gallstones and whether it acts independently of or in conjunction with ER? on inducing gallstone formation. We hypothesize that GPR30 is also involved in estrogen-dependent lithogenic actions, working independently of ER?, as both GPR30 and ER? can work through different pathways to promote the formation of estrogen- induced gallstones. This hypothesis is based on our new preliminary data showing that fed a lithogenic diet for 8 wk, ovariectomized GPR30(+/+)/ER?(-/-) mice still form gallstones in response to high doses of estrogen. By contrast, the prevalence of gallstones is significantly reduced in estrogen-treated GPR30(-/-)/ER?(-/-) mice compared to GPR30(+/+)/ER?(+/+) mice. Therefore, we plan to accomplish our goals by pursuing the following three specific aims: First, we will investigate the phenotypic characterization of GPR30 that determines susceptibility to cholesterol cholelithiasis. Second, we will study whether the activation of GPR30 leads to the lithogenesis of bile by inhibiting hepatic bile acid synthesis through the epidermal growth factor receptor (EGFR) pathway in response to high levels of estrogen. Third, we will elucidate the critical role of GPR30 in hepatic hypersecretion of biliary cholesterol and gallbladder hypomotility that accounts for rapid growth of cholesterol crystals. The proposed studies are innovative both conceptually and in the implementation of experimental approaches because distinguishing the lithogenic actions of GPR30 from those of ER? and further investigating how estrogen produces lithogenic actions through GPR30 will elucidate all the molecular mechanisms behind the formation of estrogen-induced gallstones. The planned experimental strategies are comprehensive and feasible. This project will help us gain novel mechanistic insight into the pathogenesis of estrogen-induced gallstones through GPR30 or ER? or both.